Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells.

The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 microM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 microM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 microM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.

Induction of melanoma in TPras transgenic mice.

In order to study the oncogenesis of melanocytes, transgenic mouse lines were established that express a mutated human Ha-ras (TPras) gene in pigment producing cells. The ras transgenic mice exhibit an altered phenotype, including melanocytic hyperplasia and a muted agouti coat, indicative of hyperproliferative melanocytes. These mice and their wild-type littermates have been subjected to a variety of carcinogenesis protocols, including 7, 12-dimethylbenz-[a]anthracene (DMBA), 12-O-tetradecanoylphorbol-13-acetate (TPA) and UV radiation exposure. Topical DMBA treatment of TPras mice resulted in a high incidence of melanomas. Metastatic lesions were observed in skin, lungs and lymph nodes. TPA treatment of TPras mice induced a small number of papillomas but no nevi or melanomas. UV light exposures induced papillomas in negative littermate and melanomas in some albino TPras mice. These results show that melanocytes expressing an activated Ha-ras in the TPras transgenic mice are susceptible to induction of melanoma by DMBA.

Chromosomal and genetic alterations of 7,12-dimethylbenz[a]anthracene-induced melanoma from TP-ras transgenic mice.

The TP-ras transgenic mouse line expresses an activated human T24 Ha-ras gene with a mutation in codon 12, regulated by a mouse tyrosinase promoter. The transgene is expressed in melanocytes of the skin, eyes, and brain. The mice develop cutaneous melanoma when treated with 7,12-dimethylbenz[a]anthracene. Cell lines have been generated from the cutaneous tumors and metastatic lesions. By using fluorescence in situ hybridization with mouse whole chromosome paints, the cell lines were characterized for chromosomal abnormalities. Key findings in the tumor cells included translocations of chromosome 4 and alterations in chromosome 6. One tumor cell line contained a double translocation involving chromosomes 3 and 6. To extend the results of the chromosome 4 painting, Southern analysis of the p15INK4B, p16INK4A, and p19INK4D genes was performed. Our data indicated that there were homozygous and partial allelic deletions and polymorphisms in the region of chromosome 4 containing these genes, resulting in the absence or reduced expression of the p16 product. These findings are similar to those reported for human melanoma, and the TP-ras transgenic mouse may therefore be a valuable model for studying novel strategies for melanoma prevention and treatment.